*Target (protein/gene name): Cytosolic malate dehydrogenase *NCBI Gene # or RefSeq#:15077032 *Protein ID (NP or XP #) or Wolbachia#: AAK83037.1 *Organism (including strain): Trypanosoma brucei Etiologic Risk Group (see link below):Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334 *Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Trypanosoma brucei is the protozoa that is the cause for the parasitic disease African trypanomiasis. This disease can infect both humans and animals, and is characterized by fever and headaches, until the lymphatic and circulatory systems become affected. The disease then progresses rapidly and has a variety of manifestations including severely disrupted sleep cycles due to the parasite passing through the blood brain barrier, anemia, endocrine malfunctions, cardiac problems and kidney dysfunctions. Essentiality of this protein:
However, the presence of orthologues of this gene in both T. cruzi and L. major suggests that it must fulfill some important function in trypanosomatids. http://www.sciencedirect.com/science/article/pii/S0020751905003140 Complex of proteins?: No Druggable Target: 0.8 *EC#: 1.1.1.37 Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.37 Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): -- link to Sigma (or other company) page for assay or assay reagents (substrates) http://www.sigmaaldrich.com/technical-documents/protocols/biology/_enzymatic-assay-of-malic-dehydrogenase.html -- link (or citation) to paper that contains assay information http://www.jbc.org/content/277/20/17548.full.pdf -- List cost and quantity of substrate reagents and supplier 7.3. REAGENTS 7.3.1. 100mM Potassium Phosphate Buffer, pH 7.5 at 25ºC (Buffer) $ 19.90 Prepare a 13.6 mg/mL solution in purified water using Potassium Phosphate such as Sigma-Aldrich Product Number P5379. Adjust to pH 7.5 at 25°C with 1.0 N KOH. 7.3.2. 0.14mM β-Nicotinamide Adenine Dinucleotide Solution (β-NADH) $23 Prepare a 0.14mM solution, corrected for water content, in Reagent 7.3.1 (Buffer), using β-Nicotinamide Adenine Dinucleotide Solution (reduced form), such as Sigma-Aldrich Product Number N8129. Prepare Fresh. 7.3.3. 7.6mM Oxalacetic Acid (OAA) $35.80 Immediately prior to use, prepare a 1.0 mg/mL solution in Reagent 7.3.1 (Buffer), using Oxalacetic Acid, such as Sigma-Aldrich Product Number O4126 . Product is not stable once in solution. Prepare Fresh For Each Kinetics Run. 7.3.4. Malic Dehydrogenase (MDH) $25 Immediately before use, prepare a 0.2-0.5 units/mL solution in cold Reagen 7.3.1 (Buffer). Prepare Fresh.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): msntckrvavtgaagqigysllpliaagrmlgfdqrvqlqlldispalkalegiraelmd csfplldgvvitdepkvafdkadiailcgafprkpgmerrdllqtnakifseqgrvlgev aspncrvcvvgnpantnalillreskgklnprfvtaltrldhnrataqvaerararveev knciiwgnhsgtqvpdvnsatvggkparaavdndaffdnefitivqergaeimklrglss alsaakaivdhvhdwmlgtpsgthvsmavysdgnpygvpgglifsfpvtcsggewqivsg lnvtpaiserikattteleeerrevsta *length of your protein in Amino Acids: 328
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only): *GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
*NCBI Gene # or RefSeq#:15077032
*Protein ID (NP or XP #) or Wolbachia#: AAK83037.1
*Organism (including strain): Trypanosoma brucei
Etiologic Risk Group (see link below): Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents
http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Trypanosoma brucei is the protozoa that is the cause for the parasitic disease African trypanomiasis. This disease can infect both humans and animals, and is characterized by fever and headaches, until the lymphatic and circulatory systems become affected. The disease then progresses rapidly and has a variety of manifestations including severely disrupted sleep cycles due to the parasite passing through the blood brain barrier, anemia, endocrine malfunctions, cardiac problems and kidney dysfunctions.
Essentiality of this protein:
However, the presence of orthologues of this gene in both T. cruzi and L. major suggests that it must fulfill some important function in trypanosomatids.
http://www.sciencedirect.com/science/article/pii/S0020751905003140
Complex of proteins?: No
Druggable Target: 0.8
*EC#: 1.1.1.37
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.37
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/technical-documents/protocols/biology/_enzymatic-assay-of-malic-dehydrogenase.html
-- link (or citation) to paper that contains assay information
http://www.jbc.org/content/277/20/17548.full.pdf
-- List cost and quantity of substrate reagents and supplier
7.3. REAGENTS
7.3.1. 100mM Potassium Phosphate Buffer, pH 7.5 at 25ºC (Buffer)
$ 19.90 Prepare a 13.6 mg/mL solution in purified water using Potassium Phosphate such as Sigma-Aldrich Product Number P5379. Adjust to pH 7.5 at 25°C with 1.0 N KOH.
7.3.2. 0.14mM β-Nicotinamide Adenine Dinucleotide Solution (β-NADH)
$23 Prepare a 0.14mM solution, corrected for water content, in Reagent 7.3.1 (Buffer), using β-Nicotinamide Adenine Dinucleotide Solution (reduced form), such as Sigma-Aldrich Product Number N8129. Prepare Fresh.
7.3.3. 7.6mM Oxalacetic Acid (OAA)
$35.80 Immediately prior to use, prepare a 1.0 mg/mL solution in Reagent 7.3.1 (Buffer), using Oxalacetic Acid, such as Sigma-Aldrich Product Number O4126 . Product is not stable once in solution. Prepare Fresh For Each Kinetics Run.
7.3.4. Malic Dehydrogenase (MDH)
$25 Immediately before use, prepare a 0.2-0.5 units/mL solution in cold Reagen 7.3.1 (Buffer). Prepare Fresh.
Structure Available (PDB or Homology model)
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDBignment statistics for matc
---- Query Coverage: 92%
---- Max % Identities: 51%
---- % Positives: 64%
---- Chain used for homology: 4MDH (Chain A)
Sequence ID: lcl|60447Length: 328Number of Matches: 1Related Information
Range 1: 7 to 31160447&tracks=[key:sequence_track,name:Sequence,display_name:Sequence,id:STD1,category:Sequence,annots:Sequence,ShowLabel:true][key:gene_model_track,CDSProductFeats:false][key:alignment_track,name:other%20alignments,annots:NG%20Alignments|Refseq%20Alignments|Gnomon%20Alignments|Unnamed,shown:false]&v=0:326&appname=ncbiblast&link_loc=fromHSP|GraphicsAlignment statistics for match #1||~ Score
<span style="font-size: 12px;">Query 5 RVLVTGAAGQIAYSLLYSIGNGSVFGKDQPIILVLLDITPMMGVLDGVLMELQDCALPLL 64 RV VTGAAGQI YSLL I G + G DQ + L LLDI+P + L+G+ EL DC+ PLL Sbjct 7 RVAVTGAAGQIGYSLLPLIAAGRMLGFDQRVQLQLLDISPALKALEGIRAELMDCSFPLL 66 Query 65 KDVIATDKEEIAFKDLDVAILVGSMPRRDGMERKDLLKANVKIFKCQGAALDKYAKKSVK 124 V+ TD+ ++AF D+AIL G+ PR+ GMER+DLL+ N KIF QG L + A + + Sbjct 67 DGVVITDEPKVAFDKADIAILCGAFPRKPGMERRDLLQTNAKIFSEQGRVLGEVASPNCR 126 Query 125 VIVVGNPANTNCLTASKSAPSIPKENF-SCLTRLDHNRAKAQIALKLGVTSDDVKNVIIW 183 V VVGNPANTN L + + F + LTRLDHNRA AQ+A + ++VKN IIW Sbjct 127 VCVVGNPANTNALILLRESKGKLNPRFVTALTRLDHNRATAQVAERARARVEEVKNCIIW 186 Query 184 GNHSSTQYPDVNHAKVKLQAKEVGVYEAVKDDSWLKGEFITTVQQRGAAVIKARKLSSAM 243 GNHS TQ PDVN A V + AV +D++ EFIT VQ+RGA ++K R LSSA+ Sbjct 187 GNHSGTQVPDVNSATVGGKPARA----AVDNDAFFDNEFITIVQERGAEIMKLRGLSSAL 242 Query 244 SAAKAICDHVRDIWFGTPEGEFVSMGIISDGNSYGVPDDLLYSFPVTIKDKTWKIVEGLP 303 SAAKAI DHV D GTP G VSM + SDGN YGVP L++SFPVT W+IV GL Sbjct 243 SAAKAIVDHVHDWMLGTPSGTHVSMAVYSDGNPYGVPGGLIFSFPVTCSGGEWQIVSGLN 302 Query 304 INDFSREKM 312 + E++ Sbjct 303 VTPAISERI 311 </span>Current Inhibitors: 7 hits were found in the binding database, the top hit being CHEMBL318778.
http://www.sciencedirect.com/science/article/pii/S0020751905003140
Expression Information (has it been expressed in bacterial cells): The protein is usually expressed via its natural protozoa, but has been cloned and characterized from e.coli before, pET28(a+) and pET24(a+) were used as expression vectors in bacteria.
http://www.sciencedirect.com/science/article/pii/S0020751905003140
Purification Method: Agarose gel purification the PCR products were cloned into pGEM-T-easy vector and fully sequenced
http://www.sciencedirect.com/science/article/pii/S0020751905003140
Image of protein (PyMol with features delineated and shown separately):
http://www.rcsb.org/pdb/images/4mdh_bio_r_500.jpg
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
msntckrvavtgaagqigysllpliaagrmlgfdqrvqlqlldispalkalegiraelmd csfplldgvvitdepkvafdkadiailcgafprkpgmerrdllqtnakifseqgrvlgev aspncrvcvvgnpantnalillreskgklnprfvtaltrldhnrataqvaerararveev knciiwgnhsgtqvpdvnsatvggkparaavdndaffdnefitivqergaeimklrglss alsaakaivdhvhdwmlgtpsgthvsmavysdgnpygvpgglifsfpvtcsggewqivsg
lnvtpaiserikattteleeerrevsta
*length of your protein in Amino Acids: 328
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website 35209.3
Molar Extinction coefficient of your protein at 280 nm wavelength: 21345
http://web.expasy.org/cgi-bin/protparam/protparam
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):